Immobilization of chemically and/or biologically active molecules (hereinafter referred to as biomolecules) would be greatly enhanced by the availability of a relatively inert solid-phase, polymeric substrate to which substances, such as enzymes, catalysts, hormones, lectins, drugs, vitamins, antibodies, antigens, nucleic acids, DNA and RNA segments, pesticides, dyes and fertilizers, could be readily and efficiently immobilized under mild conditions so that they would retain high activity. Unfortunately, many processes for attaching biomolecules to polymeric substrates, such as cellulose, require the utilization of relatively high temperatures, high or low pH, catalysts and initiators. These conditions and agents can often irreversibly inactivate or degrade the biomolecule, especially in the case of enzymes, antibodies, and other labile molecules. Yields are also reduced by such harsh prior art methods
The prior art teaches that the interaction of ionizing radiation with solid-phase, polymeric substrates leads to the formation of free radicals, some of which are trapped within the polymer matrix. These radicals are polymeric species, since they are formed on the polymeric substrate (e.g. cellulose). These polymeric radicals can be used to initiate a graft copolymerization reaction with hydrophilic monomers and biomolecules under conditions where such molecules can diffuse into the polymer matrix and reach the trapped radical sites.
If the monomer is present during irradiation, then irradiation will also produce radicals in the monomer phase, so that undesirable homopolymers will be formed in addition to the monomer-substrate graft. Also, if the biomolecule is present it may be damaged and deactivated by the radiation. This method of grafting to cellulose has been widely studied, and has been used to graft vinyl monomers onto paper (K. D. N. Lawrence and D. Verdin, J. Appl. Polymer Sci. 17:2653, 1973; M. U. Sadykov et al. in "Proc. Tihany Symp. Rad. Chem., 3rd Meeting," Vol. I., J. Dobo (ed.), pp. 1037, 1971; D. Campbell et al., J. Polymer Sci. 7:429, 1969). Alternatively, the cellulose may be irradiated before being brought into contact with the monomer. This pre-irradiation technique has also been applied to cellulose (F. S. Radi et al., J. Appl. Polymer Sci. 16:2685, 1972; K. D. N. Lawrence and D. Verdin, supra).
Lawrence and Verdin, supra, prepared graft copolymers of acrylamide with paper pre-irradiated at high dose rates available in electron beams in the 200-keV energy region. The temperature of the pre-irradiated cellulose was room temperature, with subsequent grafting of the acrylamide at 20.degree.-60.degree. C. One problem inherent in the Lawrence technique is that the grafting performed subsequently to the pre-irradiation is done at elevated temperatures which are hostile to most biomolecules, especially enzymes and other proteins. Thus, it would be desirable to develop a technique to maximize the copolymerization of the substrate, while providing favorable grafting conditions to allow immobilization of various biomolecules to the pre-irradiated substrate.
In 1972, Hoffman et al. (A. S. Hoffman et al., Trans. ASATO 18:10, 1972) immobilized various biomolecules, with and without extension "arms" or "leashes," onto inert polymer substrates in order to prepare blood-compatible materials. This work is covered in U.S. Pat. No. 3,826,678, issued July 30, 1974 and incorporated by reference herein. In particular, Hoffman et al. utilized mutual irradiation to graft hydroxyethyl methacrylate (HEMA) copolymers. Gombotz et al. (W. R. Gombotz et al., J. Controlled Release 2:375, 1985) and Venkataraman et al. (S. Venkataraman et al., J. Biomed. Mat. Res. 8:111, 1977) used a similar process to graft methacrylic acid (MAAc) or MAAc/HEMA copolymers. In each case, the grafted monomer was used to provide binding sites for subsequent chemical covalent binding of enzymes or drugs.
Using a different approach, some researchers have prepared polymeric gels for immobilization of enzymes and antibodies based on a co-monomer having a pendant active ester group. For example, Schnaar and Lee (R. L. Schnaar and L. C. Lee, Biochem. 14:1535, 1975) prepared the acrylic ester of N-hydroxyl succinimide (NSA), which they copolymerized with acrylamide and a cross-linker. Subsequent covalent bonding to an antibody produced an affinity gel. Adalsteinsson et al. (O. Adalsteinsson et al., J. Mol. Cat. 6:199, 1979) utilized this technique to immobilize enzymes. Lu and Feng (C. X. Lu and X. Feng, J. Poly. Sci. Chem. Edi. 18:2411, 1980) have used this technique by first preparing active ester monomers with different spacer arms, then copolymerizing them to produce a copolymer which has been used for immobilization of biomolecules.
The present invention describes a new method for immobilization of biomolecules, in which a monomer-conjugated biomolecule is grafted together with free monomer onto a hydrophilic, solid-phase polymeric substrate which has been pre-irradiated at low temperature. This method obviates the use of any initiators or catalysts which may detrimentally affect the biomolecule and reduce its activity. Advantageously, the methodology of the present invention provides for subsequent grafting of the biomolecule at very mild temperatures and pHs, and outside the radiation field, thus enhancing the activity of the immobilized biomolecule.